Herpes simplex virus type 1 (HSV-1) typically infects the oral mucosa and establishes a life-long latent infection within sensory neurons. During latency, the viral lytic genes are repressed and only one transcript is abundantly transcribed, the latency associated transcript (LAT). HSV-1 latency is characterized by intermittent episodes of recurrence during which the viral genomes present in some neurons reactivate. Virus particles are produced and transported to the original site of infection, resulting in clinical disease. Consequently, HSV-1 is responsible for significant morbidity and is the leading cause of infectious blindness in the US. While antivirals can reduce the severity of the recurrences, there is no cure. Clearly, understanding the molecular basis of how HSV regulates the lytic and latent phases of infection could provide new therapeutic approaches. Recently 8 microRNAs (miRNAs) were shown to be encoded within and adjacent to the HSV-1 LAT region. In vitro analyses have demonstrated that at least two of these miRNAs regulate HSV-1 IE gene expression. The proposed study aims to determine the roles that these miRNAs play in the pathobiology of HSV-1 infections in vivo. In order to accomplish this goal we will: 1) Construct HSV-1 recombinants containing inactivating mutations in the 8 HSV-1 miRNAs and assess these mutant viruses for changes in viral gene expression and replication in vitro; 2) Analyze these recombinants in the mouse and rabbit models for changes in replication, spread, establishment of latency and ability to reactivate. Recombinants that display altered phenotypes will be rescued and the molecular basis of the phenotypic change investigated; 3) Use the powerful photoactivatable ribonucleoside-enhanced crosslinking and immunoprecipitation (PAR-CLIP) technique to directly identify mRNA target sites occupied by HSV-1 miRNAs in cells infected with wild-type HSV-1 or with HSV-1 variants lacking specific miRNAs. Viral or cellular mRNA targets identified by these analyses will be confirmed through QT-RTPCR followed by investigation of the functional role of that gene during the viral infection cycle by knock-down and over-expression analyses, where appropriate. In order to accomplish these goals the proposed project brings together the combined expertise of the Bloom and Cullen labs. The Bloom lab at UF has expertise in the construction and characterization of HSV-1 recombinants and the study of HSV-1 pathogenesis, including latency and reactivation in the mouse and rabbit models. The Cullen lab at Duke identified the miRNAs encoded within the HSV-1 LAT region and has extensive expertise analyzing miRNA expression and function using PAR-CLIP and other assays. In total, the proposed comprehensive approach to characterize the function of the HSV-1 LAT miRNAs should provide key insights into the molecular processes that regulate the lytic and latent phases of HSV-1 infection and provide new paradigms of how miRNAs regulate herpesvirus family members in general.